Method for determining lipid bound sialic acid in plasma

Abstract

The amount of lipid bound sialic acid in a blood plasma or serum sample may be determined by an improved method which may be automated involving the following steps to be performed simultaneously on the sample and a known standard prepared from human or animal blood or tissue; diluting with distilled water; mixing; adding a mixture of a chlorinated lower aklyl alcohol; mixing, diluting with water and then treating by mixing further and centrifuging to yield a substantially clear upper phase; recovering the upper phase and adding to it a protein precipitating agent, mixing the resulting admixture; recovering the resulting precipitate, suspending the precipitate in a hydrolysis agent and determining the amount of lipid bound sialic acid present by comparing the optical density of the sample to the optical density of the standard.

What is claimed is: 
     
       1. A method of extracting lipid bound sialic acid from human blood plasma or serum and determining the amount of lipid bound sialic acid in a sample of human blood plasma or serum which comprises the following steps: a) diluting a predetermined volume of 25 microliters or less of a blood plasma or serum sample with distilled water to a volume about two times that of the predetermined volume of the sample;   b) mixing the diluted sample for a suitable period of time to obtain a substantially homogeneous sample;   c) adding to the sample a mixture of a chlorinated lower alkyl hydrocarbon and a lower alkyl alcohol, the volume of the mixture added being about forth times the predetermined volume of the blood plasma or serum sample, and the volume ratio of chlorinated hydrocarbon to alcohol in the mixture being about 2:1;   d) mixing the resulting admixture for a suitable period of time to dissolve lipid-bound sialic acid in the sample in the chlorinated hydrocarbon/alcohol mixture;   e) diluting the admixture with a buffer solution at room temperature, the volume of buffer solution being about eight times the predetermined volume of the blood plasma or serum sample;   f) mixing the diluted admixture without vortexing for a suitable period of time to obtain a substantially homogeneous admixture and centrifuging the mixture to form a substantially clear upper phase;   g) separately recovering from the clear upper phase so formed a predetermined volume of the upper phase;   h) adding to the predetermined volume of the upper phase an amount of a mixture of a protein-precipitating agent and water without any adsorbing material, the amount of mixture being effective to cause precipitation of the lipid bound sialic acid;   i) mixing the resulting admixture;   j) separately recovering the resulting precipitate;   k) suspending the precipitate in a hydrolysis agent;   l) determining the amount of lipid bound sialic acid present in the suspended precipitate and thereby the amount present in the blood plasma or serum sample.   
     
     
       2. A method according to claim 1, wherein in step (b) the mixing takes place for at least 2 seconds. 
     
     
       3. A method according to claim 1, wherein in step (c) the volume of the added mixture is about 1 ml. 
     
     
       4. A method according to claim 1, wherein in step (c) the lower alkyl alcohol is methanol, ethanol, propanol, n-butanol, isopropanol, isobutanol or isoamyl alcohol. 
     
     
       5. A method according to claim 4, wherein in step (c) the lower alkyl alcohol is methanol. 
     
     
       6. A method according to claim 1, wherein in step (c) the chlorinated lower alkyl hydrocarbon is chloroform. 
     
     
       7. A method according to claim 1, wherein in step (d) the mixing takes place for at least 10 seconds. 
     
     
       8. A method according to claim 1, wherein in step (a) the volume of water added is about 0.2 ml. 
     
     
       9. A method according to claim 1, wherein in step (f) the mixing comprises gentle inversion for at least 30 seconds followed by centrifuging at above 3500 rpm for at least 5 minutes. 
     
     
       10. A method according to claim 1, wherein in step (g) the separately recovering comprises removing the upper phase from the lower phase. 
     
     
       11. A method according to claim 1, wherein in step (g) the predetermined amount of the upper phase is about 0.2 ml. 
     
     
       12. A method according to claim 1, wherein in step (h) the protein-precipitating agent is phosphotungstic aoid, trichloroacetic acid, ammonium sulfate or a mixture thereof. 
     
     
       13. A method according to claim 1, wherein in step (h) the mixture comprises 25 ml of phosphotungstic acid and distilled water in a volume ratio of 1:3. 
     
     
       14. A method according to claim 1, wherein in step (i) the mixing takes place for at least 3 seconds. 
     
     
       15. A method according to claim 1, wherein in step (j) the separately recovering comprises centrifuging for at least 3 minutes at a speed above about 1500 rpm. 
     
     
       16. A method according to claim 11, wherein in step (k) the hydrolysis agent is resorcinol. 
     
     
       17. A method according to claim wherein step (l) the amount of lipid bound sialic acid is determined by adding to the suspended precipitate a volume of resorcinol reagent, mixing, boiling for 15 minutes, cooling for about 5 minutes in cold water, adding a mixture of butylacetate and n-butanol (85:15 v/v) in a volume about twice said volume of resorcinol reagent mixing, centrifuging for about 5 minutes at above 2500 rpm, separating the organic layer, reading at 580 nm the extracted blue color present in the organic layer, determining the amount of lipid bound sialic acid by comparing the reading obtained at 580 nm to that obtained for a standard having a known amount of lipid bound sialic acid and applying the formula: ##EQU2## Where A=the concentration of the standard, B=the optical density of the sample and C=the optical density of the standard. 
     
     
       18. A method according to claim 17, wherein the known standard is tested exactly the same as, and simultaneously with, the sample in accord with the method of claim 1. 
     
     
       19. A method according to claim 18, wherein the standard is animal blood plasma or serum, human blood plasma or serum or an extract of human or animal tissue. 
     
     
       20. A method according to claim 17, wherein the volume of resorcinol reagent is about 0.5 ml. 
     
     
       21. A method of diagnosing cancer in a human subject which comprises determining the amount of lipid bound sialic acid in a sample of the subject's blood plasma or serum according to the method of claim 1 and comparing the amount so determined with values obtained for subjects known to have cancer. 
     
     
       22. A method of diagnosing cancer in a human subject which comprises determining at regular time intervals the amount of lipid bound sialic acid in a sample of the subject's blood plasma or serum according to the method of claim 1 and comparing the amounts so determined with amounts previously obtained for the subject. 
     
     
       23. A method according to claim 1, wherein the amount of lipid bound sialic acid determined in the blood plasma or serum sample is compared with a standard having a known concentration of lipid bound sialic acid which has been tested at the same time and by the same method as the sample with the effect that discrepancies in the test results attributable to variations in the techniques used in the test of the sample and the standard are eliminated.